Opioid activity is a function of its receptor steady state level. How neuronal cells regulate the opioid receptor level could be the key in understanding the cellular mechanism of opioid tolerance. Prior to the cloning of the three opioid receptor types, reagents such as receptor specific antibodies are not available for such studies. With the development of receptor specific antibodies and epitope tagged opioid receptor, we and other laboratories have demonstrated that delta-opioid receptor internalized via the clathrin-coated vesicles and trafficked to the endosomes and later to lysosomes for degradation. This iternary of opioid receptor is similar to that observed with other G protein-coupled receptors (GPCR). The exact cellular components involved and the signals that triggered the opioid receptor internalization remain elusive. Therefore, in the proposed studies, we will determine the beta-opioid receptor domains that are involved in the receptor internalization and down-regulation, and the cellular proteins that participate in the trafficking of delta-opioid receptor. The approaches we will use is to construct delta-opioid receptor mutants, i.e., truncation mutations to locate the general area followed by site-directed mutagenesis to pin-point the amino acids involved. Since earlier reports with GPCR and delta-opioid receptor suggested involvement of protein kinases in receptor internalization and down-regulation, initial focus will be on the Ser and Thr within the intracellular domains of the receptor that can be phosphorylated. However, the random mutation approach will also be used to identify any motifs or amino acids that might be involved and are not predicted by our current understanding of GPCR. IN order to accomplish this goal, a green-fluorescent protein-delta receptor conjugate will de developed. The receptor domains identified to be involved in receptor down-regulation and desensitization will be used to isolate the cellular proteins that participate in the receptor trafficking. The comparison of the immunoprecipitated complexes between wild-type and mutant receptors with antibodies against proteins that are known to participate in protein trafficking, e.g., G proteins, the roles of these proteins in receptor internalization and down-regulation will be identified. These information in combination with the isolation of receptor complexes in the Tet-on system in which the receptor levels can be induced will allow us to deduce and isolate other cellular proteins that are involved in this processes. In addition, we will continue to explore the mechanism in which the opioid receptor mRNA level can be regulated by second messengers and the role of the mRNA levels on the steady state level of receptor protein.